Helper oligonucleotide for improved efficiency of amplification and detection/quantitation of nucleic acids

ABSTRACT

Improved methods for the detection and quantitation of a target nucleic acid in a sample using a non-extending helper oligonucleotide are described. The methods include contacting nucleic acids in a sample with amplification reagents including one or more primers, one or more non-extending helper oligonucleotides, and one or more probes. The non-extending helper oligonucleotide facilitates and increases the target nucleic acid accessibility of one or more of the primers, result in greater accumulation of amplicon production, thereby increasing the efficiency and sensitivity of the amplification assay, including amplification assays for Hepatitis C Virus (HCV), for example, HCV Genotype 5. Kits, articles of manufacture, and reaction mixtures are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/370,049, filed Aug. 2, 2016, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to the field of in vitro diagnostics.Within this field, the present invention is directed to improved methodsfor the detection of a target nucleic acid that may be present in asample (e.g., biological or non-biological sample). In particular, thepresent invention concerns the detection and quantitation of a targetnucleic acid, with the aid or help of a non-extending helperoligonucleotide that enhances the activity of the primers. By enhancingthe activity of the primers, the non-extending helper oligonucleotideimproves amplification efficiency, which results in greater and enhancedamplicon production. The improvement, therefore, allows for a moreefficient and more sensitive detection and quantitation method. Theinvention further provides reaction mixtures and kits containing thenon-extending helper oligonucleotide, primers, and probes for detectionand quantitation target nucleic acid that may be present in a sample.This present invention is useful, for example, for the detection andquantitation of viral or bacterial nucleic acid in samples.

BACKGROUND OF THE INVENTION

In the field of molecular diagnostics, the amplification anddetection/quantitation of nucleic acids is of considerable significanceand importance. Examples for diagnostic applications of nucleic acidamplification and detection are for the detection and amplification ofmicrobial nucleic acids. Such microbial nucleic acids may includebacterial nucleic acids and/or viral nucleic acids. The amplificationand detection/quantitation techniques are suitable for viral nucleicacid targets, such as Human Papilloma Virus (HPV) or West Nile Virus(WNV), or the routine screening of blood donations for the presence ofHuman Immunodeficiency Virus (HIV), Hepatitis A Virus (HAV), Hepatitis BVirus (HBV), or Hepatitis C Virus (HCV). The amplification anddetection/quantitation techniques are also suitable for bacterialnucleic acid targets or the analysis of oncology markers or the like.

The most prominent and widely used method for amplification (anddetection/quantitation) of nucleic acid targets is the Polymerase ChainReaction (PCR). Other amplification techniques include Ligase ChainReaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair ChainReaction, 3 SR, NASBA, Strand Displacement Amplification (SDA),Transcription Mediated Amplification (TMA), and Qβ-amplification.

Automated systems for PCR-based analysis often make use of a real-timedetection of product amplification during the PCR process in the samereaction vessel. Key to such methods is the use of modifiedoligonucleotides carrying reporter groups or labels. PCR utilizes apolymerase enzyme (U.S. Pat. Nos. 4,683,195 and 4,683,202). Relatedsignificant improvements are, e.g., real-time detection of amplifiedproducts during PCR utilizing modified oligonucleotides carryingreporter groups or labels known as hydrolization or 5′-nuclease probessuch as used in commercial assays on COBAS® TaqMan® (U.S. Pat. Nos.5,210,015 and 5,487,972). Other improved amplification and detectionmethods are known as Molecular Beacons technology (International PatentPublication No. WO 95/13399) or methods utilizing an oligonucleotidecomprising a minor groove binder (MGB) portion (International PatentPublication Nos. WO 03/062445 and WO 2006/135765). It is further knownthat the use of primers containing an added oligonucleotide with a highGC content at the 5′ terminus of at least one of these primers displaysan improvement in amplification efficiency (Liu, et al., Genome Research7:389-398 (1997); International Patent Publication No. WO 01/94638; andU.S. Patent Publication No. US 2004/0110182). The final quantity of theamplified product after approximately 12 to 40 cycles of PCR is markedlyhigher for primers to which, e.g., a GGAC unit has been added to the 5′termini than for the unmodified primers.

Afonina, et al., BioTechniques 43(3):1-3 (2007); International PatentPublication No. WO 2006/135765) describe the increase of real-time PCRfluorescent signal and thereby obtaining improved amplificationefficiency by using primers with short adenine and thymine rich flaps,scattered randomly, at the 5′ terminus and minor groove binder (MGB)fluorescent hybridization probes.

Similarly, Babiel, et al. (U.S. Pat. No. 9,447,476) describe how theaddition of polyN to primers can result in the reduction or suppressionof the formation of unwanted high molecular weight products, therebyavoiding false-negative or false-positive results.

Thus, there is always a need in the art for improvements on existingmethods. For example, there is a need in the art to provide a method forsimple and reliable detection and quantitation of a nucleic acid target.There is, in particular a need for improving the efficiency of DNAamplification and detection/quantitation.

SUMMARY OF THE INVENTION

Certain embodiments in the present disclosure relate to new methods anduses for amplification, detection, and quantitation of target nucleicacid in a sample (e.g., a biological or non-biological sample). Forexample, certain embodiments of the present disclosure relate tosingleplex or multiplex detection and quantitating of a microbe, such asvirus (e.g., HPV, WNV, HAV, HBV, HCV, and HIV) by a real-time polymerasechain reaction (PCR) in a single test tube or reaction vessel. Theimprovement is based on the use of a non-extending helperoligonucleotide in an amplification reaction, such as PCR. It isbelieved that the non-extending helper oligonucleotide acts as a sort ofa helper oligonucleotide or a helper primer, in that it facilitates andenhances amplification that is mediated by the (extending) primers. Itis further believed that the presence of the non-extending helperoligonucleotide reduces or lowers the Gibbs Free Energy of the secondarystructure of the target region of the target nucleic acid. In somecases, the non-extending helper oligonucleotide is believed toreduce/lower the Gibbs Free energy of the secondary structure of theprimer-binding region of the target nucleic acid. The non-extendinghelper oligonucleotide may anneal within the same region as one or moreof the primers. However, the non-extending helper oligonucleotide doesnot extend, in contrast to the one or more primers. That is, thenon-extending helper oligonucleotide does not extend to generate anamplicon. Instead of extending, the non-extending helper oligonucleotidefacilitates and increases the target accessibility of the primer orprimers, which do extend and generate amplicons. That is, like theextending primer, the non-extending helper oligonucleotide anneals tothe target nucleic acid, but unlike the extending primer, thenon-extending helper oligonucleotide does not extend. There are manyways to prevent an oligonucleotide from extending, which are and wouldbe well known by one of ordinary skill in the art. For example, theaddition of poly(A) sequences to the 3′-end of the oligonucleotideprevents extension. It is further believed that the poly(A) sequences donot likely anneal to any portion of the target nucleic acid, whichthereby destabilizes the interaction between the non-extending helperoligonucleotide and the target nucleic acid, which in turn, opens up thesecondary structure and allowing for displacement of the non-extendinghelper oligonucleotide with the (extending) primer. Additionally,replacing the 3′-OH group with a phosphate group (i.e., phosphorylation)also prevents extension. It is believed that the non-extending helperoligonucleotide assists the amplification by reducing the Gibbs FreeEnergy of the secondary structure in the primer binding region of thetarget nucleic acid, thereby improving the accessibility of one or moreof the primers that extend (see, FIGS. 2 and 4). This results in animprovement in PCR amplification, such that more PCR product (i.e.,amplicon) accumulates in the presence of the non-extending helperoligonucleotide than in the absence of the non-extending helperoligonucleotide (see, FIGS. 1 and 3). That is, the overall efficiency ofthe PCR assay is improved and/or enhanced by the non-extending helperoligonucleotide. Because the efficiency of the PCR assay is improved, itis possible to successfully amplify even small amounts of startingmaterial (i.e., low copy numbers), in the presence of the non-extendinghelper oligonucleotide. Thus, the non-extending helper oligonucleotidealso increases the sensitivity of the assay.

Embodiments include methods of detection and quantitation of a targetnucleic acid comprising performing at least one cycling step, which mayinclude an amplifying step and a hybridizing step. Furthermore,embodiments include primers, probes, and one or more non-extendinghelper oligonucleotides, and kits that are designed for the detectionand quantitation of a target nucleic acid in a single reaction vessel ortube.

The present disclosure also provides for methods of detecting thepresence or absence of a target nucleic acid in a biological sample froman individual. These methods can be employed to detect the presence orabsence of a target nucleic acid in plasma, for use in blood screeningand diagnostic testing. Additionally, the same test may be used bysomeone experienced in the art to assess urine and other sample types todetect and/or quantitate a target nucleic acid. Such methods generallyinclude performing at least one cycling step, which includes anamplifying step and a dye-binding step. Typically, the amplifying stepincludes contacting the sample with a plurality of pairs ofoligonucleotide primers and, in this case, one or more non-extendinghelper oligonucleotides, to produce one or more amplification productsif a nucleic acid molecule is present in the sample, and the dye-bindingstep includes contacting the amplification product with adouble-stranded DNA binding dye. Such methods also include detecting thepresence or absence of binding of the double-stranded DNA binding dyeinto the amplification product, wherein the presence of binding isindicative of the presence of a target nucleic acid in the sample, andwherein the absence of binding is indicative of the absence of targetnucleic acid in the sample. A representative double-stranded DNA bindingdye is ethidium bromide. Other nucleic acid-binding dyes include DAPI,Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes suchas YO-YO® and SYBR® Green. In addition, such methods also can includedetermining the melting temperature between the amplification productand the double-stranded DNA binding dye, wherein the melting temperatureconfirms the presence or absence of a nucleic acid nucleic acid.

In a further embodiment, a kit for detecting and/or quantitating one ormore target nucleic acids is provided. The kit can include one or moresets of primers specific for amplification of the target nucleic acid;one or more non-extending helper oligonucleotide; and one or moredetectable oligonucleotide probes specific for detection of theamplification products.

In one aspect, the kit can include probes already labeled with donor andcorresponding acceptor moieties, e.g., another fluorescent moiety or adark quencher, or can include fluorophoric moieties for labeling theprobes. The kit can also include nucleoside triphosphates, nucleic acidpolymerase, and buffers necessary for the function of the nucleic acidpolymerase. The kit can also include a package insert and instructionsfor using the primers, non-extending helper oligonucleotides, probes,and fluorophoric moieties to detect the presence or absence ofamplification products/target nucleic acids in a sample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present subject matter, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows real-time PCR growth curves of an experiment showing theprimers, probes, and non-extending helper oligonucleotides and probesspecific for a target nucleic acid (of HCV).

FIG. 2 shows the predicted secondary structure of the (HCV) targetregion in the presence of the non-extending helper oligonucleotide,showing a ΔG of −107.2 kcal/mol.

FIG. 3 shows real-time PCR growth curves of an experiment showing theprimers, probes, and non-extending helper oligonucleotides and probesspecific for a target nucleic acid (of HCV).

FIG. 4 shows the predicted secondary structure of the (HCV) targetregion in the presence of the non-extending helper oligonucleotide,showing a ΔG of −98.6 kcal/mol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to new and improved methods and uses foramplifying, detecting, and quantitating a nucleic acid target that maybe present in a sample (e.g., a biological sample) comprising at least aprimer pair for generating an amplicon, a detectable probe specific forthe amplicon, and a non-extending helper oligonucleotide. Theimprovement is, in particular, based on the fact that the amplification(i.e., generation of the amplicon) is enhanced and increased in thepresence of a non-extending helper oligonucleotide. That is, thenon-extending helper oligonucleotide enhances and improves the activityand efficiency of one or more of the primers that extends, therebyimproving and increasing the efficiency of the PCR reaction. This hasthe effect of increasing the sensitivity of the assay. This presentinvention can be used for any number of applications, including, but notlimited to the amplification, and detection/quantitation of microbialnucleic acids (e.g., viral or bacterial nucleic acid). Examples ofviruses for use with the present invention include HPV, WNV, HAV, HBV,HCV, and HIV.

Diagnosis of a microbial (e.g., viral or bacterial) infection by nucleicacid amplification provides a method for rapidly, accurately, reliably,specifically, and sensitively detecting and/or quantitating theinfection. A real-time reverse-transcriptase PCR assay for detectingand/or quantitating microbial nucleic acids in a non-biological orbiological sample is described herein. Primers and probes for detectingand/or quantitating target nucleic acid (such as microbial nucleic acid,like viral or bacterial nucleic acid) are provided, as are articles ofmanufacture or kits containing such primers and probes. The increasedspecificity and sensitivity of real-time PCR for detection of targetnucleic acid compared to other methods, as well as the improved featuresof real-time PCR including sample containment and real-time detectionand quantitating of the amplified product, make feasible theimplementation of this technology for routine diagnosis of microbialinfections (e.g., viral or bacterial infections) in the clinicallaboratory. Additionally, this technology may be employed for bloodscreening as well as for prognosis. Such a detection assay may also bemultiplexed with other assays for the detection of other target nucleicacids (e.g., other microbial nucleic acids, or other/different genotypesof the same microbe), in parallel.

The present disclosure includes, by way of example, oligonucleotideprimers, non-extending helper oligonucleotides, and fluorescent labeledhydrolysis probes that hybridize to the HCV genome, in order tospecifically identify HCV using, e.g., TaqMan® detection technology.

The term “primer(s)” or “extending primer(s)” as used herein is known tothose skilled in the art and refers to oligomeric compounds, primarilyto oligonucleotides but also to modified oligonucleotides that are ableto “prime” DNA synthesis by a template-dependent DNA polymerase, i.e.,the 3′-end of the, e.g., oligonucleotide provides a free 3′-OH groupwhere further “nucleotides” may be attached by a template-dependent DNApolymerase establishing 3′ to 5′ phosphodiester linkage wherebydeoxynucleoside triphosphates are used and whereby pyrophosphate isreleased.

Specifically, the disclosed methods may include performing at least onecycling step that includes amplifying one or more portions of thenucleic acid molecule gene target from a sample using one or more pairsof primers. “Primer(s)” or “extending primer(s)” as used herein refer tooligonucleotide primers that specifically anneal to nucleic acidsequences found in the target region in the sample, and initiate DNAsynthesis therefrom under appropriate conditions producing therespective amplification products. The amplification product shouldcontain the nucleic acid sequences that are complementary to one or moredetectable probes specific for the target nucleic acid. “Probe(s)” or“detectable probe(s)” as used herein refer to oligonucleotide probesthat specifically anneal to nucleic acid sequences found in targetregion in the sample. Each cycling step includes an amplification step,a hybridization step, and a detection step, in which the sample iscontacted with the one or more detectable probes for detection of thepresence or absence of target nucleic acid in the target region of thesample.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., nucleic acidmolecules from a microbe, such as virus or bacteria). Amplifying anucleic acid molecule typically includes denaturing the template nucleicacid, annealing primers to the template nucleic acid at a temperaturethat is below the melting temperatures of the primers, and enzymaticallyelongating from the primers to generate an amplification product.Amplification typically requires the presence of deoxyribonucleosidetriphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and anappropriate buffer and/or co-factors for optimal activity of thepolymerase enzyme (e.g., MgCl₂ and/or KCl).

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. “Hybridization conditions” typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ nuclease activity” refers to an activity of a nucleicacid polymerase, typically associated with the nucleic acid strandsynthesis, whereby nucleotides are removed from the 5′ end of nucleicacid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished, if necessary.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., at-butyl benzyl, a phosphate, a C5-methyl-dC, a C5-ethyl-dC, aC5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, aC5-propynyl-dU, a C7-propynyl-dA, a C7-propynyl-dG, aC5-propargylamino-dC, a C5-propargylamino-dU, a C7-propargylamino-dA, aC7-propargylamino-dG, a 7-deaza-2-deoxyxanthosine, a pyrazolopyrimidineanalog, a pseudo-dU, a nitro pyrrole, a nitro indole, 2′-0-methylribo-U, 2′-0-methyl ribo-C, an N4-ethyl-dC, an N6-methyl-dA, a5-propynyl dU, a 5-propynyl dC, and the like. Many other modifiednucleotides that can be substituted in the oligonucleotides are referredto herein or are otherwise known in the art. In certain embodiments,modified nucleotide substitutions modify melting temperatures (Tm) ofthe oligonucleotides relative to the melting temperatures ofcorresponding unmodified oligonucleotides. To further illustrate,certain modified nucleotide substitutions can reduce non-specificnucleic acid amplification (e.g., minimize primer dimer formation or thelike), increase the yield of an intended target amplicon, and/or thelike in some embodiments. Examples of these types of nucleic acidmodifications are described in, e.g., U.S. Pat. No. 6,001,611, which isincorporated herein by reference. Other modified nucleotidesubstitutions may alter the stability of the oligonucleotide, or provideother desirable features.

Detection of Target Nucleic Acid

The present disclosure provides methods to detect and quantitate atarget nucleic acid in a sample by amplifying a portion of the targetnucleic acid sequence. By way of example, provided are methods to detectand quantitate HCV in a sample by amplifying a portion of the HCVnucleic acid sequence. Specifically, primers, non-extending helperoligonucleotides, and probes to amplify and detect/quantitate HCVnucleic acid molecule targets are provided by the embodiments in thepresent disclosure.

For amplification, detection, and quantitation of HCV, primers,non-extending helper oligonucleotides, and probes are provided. HCVnucleic acids other than those exemplified herein can also be used todetect HCV in a sample. For example, functional variants can beevaluated for specificity and/or sensitivity by those of skill in theart using routine methods. Representative functional variants caninclude, e.g., one or more deletions, insertions, and/or substitutionsin the HCV nucleic acids disclosed herein.

More specifically, embodiments of the oligonucleotides each include anucleic acid with a sequence selected from SEQ ID NOs:1-5, asubstantially identical variant thereof in which the variant has atleast, e.g., 80%, 90%, or 95% sequence identity to one of SEQ IDNOs:1-5, or a complement of SEQ ID NOs:1-5 and the variant. The forwardprimer, reverse primers, and detectable probes (SEQ ID NOs:1-4) are froma commercially available HCV genotyping assay (Cobas® HCV GT for usewith the Cobas® 4800 system, Roche).

TABLE 1 HCV Oligonucleotides SEQ Oligo Type Oligo Name ID NO: SequenceModifications Forward AYHCV500 1 TGGGCAGGGTGGTTGCTK K: t-Butyl Benzyl-dCPrimer 1TBB Reverse AYHCV500 2 GTTGCATAGTTTACCCCGTCCTCAJJ: t-Butyl Benzyl-dA Primer 2TBB Reverse AYHCV501 3GTTGCATAGTTTATCCCGTCCTCAJ J: t-Butyl Benzyl-dA Primer 2BB DetectableAYHCV501 4 EATCCCGQCTCGTAGGCGGCCCCGTTGP Q: BHQ-2 Probe 2HBH6P: Phosphate E: Threo-HEX Non-Extending RM_111 5GTTGCATAGTTTATCCCGTCTTCAAGAA Helper Oligo CCTTCACACCGTGTGCGAAAAAAAA

In one embodiment, the above described sets of HCV primers,non-extending helper oligonucleotides, and probes are used in order toprovide for detection of HCV in a biological sample suspected ofcontaining HCV (Table 1). The sets of primers, non-extending helperoligonucleotides, and probes may comprise or consist of the primers,non-extending helper oligonucleotide, and probes specific for the HCVnucleic acid sequences, comprising or consisting of the nucleic acidsequences of SEQ ID NOs:1-5. In another embodiment, the primers,non-extending helper oligonucleotides, and probes for the HCV targetcomprise or consist of a functionally active variant of any of theprimers, non-extending helper oligonucleotides, and probes of SEQ IDNOs:1-5.

A functionally active variant of any of the primers, non-extendinghelper oligonucleotides, and/or probes of SEQ ID NOs:1-5 may beidentified by using the primers, non-extending helper oligonucleotides,and/or probes in the disclosed methods. A functionally active variant ofa primer, non-extending helper oligonucleotides, and/or probe of any ofthe SEQ ID NOs:1-5 pertains to a primer, non-extending helperoligonucleotides, and/or probe which provide a similar or higherspecificity and sensitivity in the described method or kit as comparedto the respective sequence of SEQ ID NOs:1-5.

The variant may, e.g., vary from the sequence of SEQ ID NOs:1-5 by oneor more nucleotide additions, deletions or substitutions such as one ormore nucleotide additions, deletions or substitutions at the 5′ endand/or the 3′ end of the respective sequence of SEQ ID NOs:1-5. Asdetailed above, a primer, non-extending helper oligonucleotide, and/orprobe may be chemically modified, i.e., a primer, non-extending helperoligonucleotide, and/or probe may comprise a modified nucleotide or anon-nucleotide compound. A primer, non-extending helper oligonucleotide,and/or probe is then a modified oligonucleotide. “Modified nucleotides”(or “nucleotide analogs”) differ from a natural “nucleotide” by somemodification but still consist of a base or base-like compound, apentofuranosyl sugar or a pentofuranosyl sugar-like compound, aphosphate portion or phosphate-like portion, or combinations thereof.For example, a “label” may be attached to the base portion of a“nucleotide” whereby a “modified nucleotide” is obtained. A natural basein a “nucleotide” may also be replaced by, e.g., a 7-desazapurinewhereby a “modified nucleotide” is obtained as well. The terms “modifiednucleotide” or “nucleotide analog” are used interchangeably in thepresent application. A “modified nucleoside” (or “nucleoside analog”)differs from a natural nucleoside by some modification in the manner asoutlined above for a “modified nucleotide” (or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify a nucleic acid molecule corresponding to a targetregion. Oligonucleotides corresponding to, e.g., alternative portions ofthe target can be designed using, for example, a computer program suchas OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Importantfeatures when designing oligonucleotides to be used as amplificationprimers include, but are not limited to, an appropriate sizeamplification product to facilitate detection (e.g., byelectrophoresis), similar melting temperatures for the members of a pairof primers, and the length of each primer (i.e., the primers need to belong enough to anneal with sequence-specificity and to initiatesynthesis but not so long that fidelity is reduced duringoligonucleotide synthesis). Typically, oligonucleotide primers are 8 to50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides inlength).

Oligonucleotides of the present invention include “helperoligonucleotide(s)” or “non-extending helper oligonucleotide(s).” Insome cases, the non-extending helper oligonucleotide will anneal, inpart, to a portion within the target nucleic acid. Because thenon-extending helper oligonucleotides is believed to facilitate accessof the extending primer or primers to the target nucleic acid, thenon-extending helper oligonucleotide may act on or near the samelocation as where the extending primer will anneal. Therefore, in someinstances, the non-extending helper oligonucleotide will anneal, in partor in whole, to the same region within the target nucleic acid as theextending primer. That is, in some cases, the non-extending helperoligonucleotide and the extending primer will anneal to the sameportion, in part or in whole, within the target nucleic acid. Thus, insome cases, the non-extending helper oligonucleotide and the extendingprimer may share the same sequences, in part or in whole. It is believedthat the non-extending helper oligonucleotide lowers the Gibbs freeenergy of the secondary structure of the target nucleic acid. This thenfacilitates annealing of one or more extending primers to the target,which would then initiate extension via DNA polymerases (i.e., nucleicacid synthesis). Thus, the non-extending helper oligonucleotide

In order to prevent the helper oligonucleotide itself from extending,any number of modifications may be made to the helper oligonucleotide.Such modifications would be well known to one of ordinary skill in theart. Examples of such modifications include the addition of poly(A)nucleotides to the 3′-terminus of the helper oligonucleotide, and/or byreplacing the 3′-OH group with a phosphate group (i.e.,phosphorylation). It is believed that the addition of poly(A) sequences(which will not likely anneal to the target nucleic acid) destabilizesthe interaction between the non-extending helper oligonucleotide and thetarget nucleic acid, thereby opening up the secondary structure, whichfacilitates access of the target nucleic acid region to the (extending)primer.

In addition to a set of primers, the methods may use one or more probesin order to detect the presence or absence of a target nucleic acid. Theterm “probe” refers to synthetically or biologically produced nucleicacids (DNA or RNA), which by design or selection, contain specificnucleotide sequences that allow them to hybridize, under definedpredetermined stringencies specifically (i.e., preferentially), to“target nucleic acids.” A “probe” can be referred to as a “detectionprobe” meaning that it detects the target nucleic acid.

In some embodiments, the probes can be labeled with at least onefluorescent label. In one embodiment, the probes can be labeled with adonor fluorescent moiety, e.g., a fluorescent dye, and a correspondingacceptor moiety, e.g., a quencher. In one embodiment, the probecomprises or consists of a fluorescent moiety and the nucleic acidsequences comprise or consist of SEQ ID NO:4.

Designing oligonucleotides to be used as probes can be performed in amanner similar to the design of primers. Embodiments may use a singleprobe or a pair of probes for detection of the amplification product.Depending on the embodiment, the probe(s) use may comprise at least onelabel and/or at least one quencher moiety. As with the primers, theprobes usually have similar melting temperatures, and the length of eachprobe must be sufficient for sequence-specific hybridization to occurbut not so long that fidelity is reduced during synthesis.Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22,23, 24, or 25) nucleotides in length.

Constructs can include vectors each containing one primers,non-extending helper oligonucleotides, and probes nucleic acidmolecules. For example, constructs can include vectors each containingone of microbial target primers, non-extending helper oligonucleotides,and probes nucleic acid molecules (e.g., SEQ ID NOs:1, 2, 3, 4, and 5,for HCV). Constructs can be used, for example, as control templatenucleic acid molecules. Vectors suitable for use are commerciallyavailable and/or produced by recombinant nucleic acid technology methodsroutine in the art. Nucleic acid molecules can be obtained, for example,by chemical synthesis, direct cloning from target region, or by nucleicacid amplification.

Constructs suitable for use in the methods typically include sequencesencoding a selectable marker (e.g., an antibiotic resistance gene) forselecting desired constructs and/or transformants, and an origin ofreplication. The choice of vector systems usually depends upon severalfactors, including, but not limited to, the choice of host cells,replication efficiency, selectability, inducibility, and the ease ofrecovery.

Constructs containing the target nucleic acid molecules can bepropagated in a host cell. As used herein, the term host cell is meantto include prokaryotes and eukaryotes such as yeast, plant and animalcells. Prokaryotic hosts may include E. coli, Salmonella typhimurium,Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts includeyeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cellssuch as COS cells or Chinese hamster ovary (CHO) cells, insect cells,and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. Aconstruct can be introduced into a host cell using any of the techniquescommonly known to those of ordinary skill in the art. For example,calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transferare common methods for introducing nucleic acids into host cells. Inaddition, naked DNA can be delivered directly to cells (see, e.g., U.S.Pat. Nos. 5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). These two oligonucleotide primers are typically a forward primerand a reverse primer. Primers useful in some embodiments includeoligonucleotides capable of acting as points of initiation of nucleicacid synthesis within the described HCV nucleic acid sequences (e.g.,SEQ ID NOs:1, 2, and 3). A primer can be purified from a restrictiondigest by conventional methods, or it can be produced synthetically. Theprimer is preferably single-stranded for maximum efficiency inamplification, but the primer can be double-stranded. Double-strandedprimers are first denatured, i.e., treated to separate the strands. Onemethod of denaturing double stranded nucleic acids is by heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence. The temperature forannealing is usually from about 35° C. to about 65° C. (e.g., about 40°C. to about 60° C.; about 45° C. to about 50° C.). Annealing times canbe from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec;about 30 sec to about 40 sec). The reaction mixture is then adjusted toa temperature at which the activity of the polymerase is promoted oroptimized, i.e., a temperature sufficient for extension to occur fromthe annealed primer to generate products complementary to the templatenucleic acid. The temperature should be sufficient to synthesize anextension product from each primer that is annealed to a nucleic acidtemplate, but should not be so high as to denature an extension productfrom its complementary template (e.g., the temperature for extensiongenerally ranges from about 40° C. to about 80° C. (e.g., about 50° C.to about 70° C.; about 60° C.). Extension times can be from about 10 secto about 5 min (e.g., about 30 sec to about 4 min; about 1 min to about3 min; about 1 min 30 sec to about 2 min).

The genome of a retrovirus or RNA virus (e.g., HCV as well as otherflaviviruses), is comprised of a ribonucleic acid, i.e., RNA. In suchcase, the template nucleic acid, RNA, must first be transcribed intocomplementary DNA (cDNA) via the action of the enzyme reversetranscriptase. Reverse transcriptases use an RNA template and a shortprimer complementary to the 3′ end of the RNA to direct synthesis of thefirst strand cDNA, which can then be used directly as a template forpolymerase chain reaction.

PCR assays can employ target nucleic acid such as RNA or DNA (cDNA). Thetemplate nucleic acid need not be purified; it may be a minor fractionof a complex mixture, such as microbial target nucleic acid contained inhuman cells. Microbial nucleic acid molecules, such as from viruses,such as HPV, WNV, HAV, HBV, HCV, and HIV, may be extracted from abiological sample by routine techniques such as those described inDiagnostic Molecular Microbiology: Principles and Applications (Persinget al. (eds), 1993, American Society for Microbiology, Washington D.C.).Nucleic acids can be obtained from any number of sources, such asplasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

The oligonucleotide primers (e.g., SEQ ID NOs:1-3 for HCV) are combinedwith PCR reagents under reaction conditions that induce primerextension. For example, chain extension reactions generally include 50mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin,0.5-1.0 μg denatured template DNA, 50 pmoles of each oligonucleotideprimer, 2.5 U of Taq polymerase, and 10% DMSO). The reactions usuallycontain 150 to 320 μM each of dATP, dCTP, dTTP, dGTP, or one or moreanalogs thereof.

The newly-synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target nucleic acid molecules. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength. In certain systems, non-fluorescent energy can betransferred between donor and acceptor moieties, by way of biomoleculesthat include substantially non-fluorescent donor moieties (see, forexample, U.S. Pat. No. 7,741,467).

In one example, an oligonucleotide probe can contain a donor fluorescentmoiety (e.g., FAM) and a corresponding quencher (e.g., BlackHoleQuenchers™ (BHQ) (such as BHQ2)), which may or not be fluorescent, andwhich dissipates the transferred energy in a form other than light. Whenthe probe is intact, energy transfer typically occurs between the donorand acceptor moieties such that fluorescent emission from the donorfluorescent moiety is quenched the acceptor moiety. During an extensionstep of a polymerase chain reaction, a probe bound to an amplificationproduct is cleaved by the 5′ to 3′ nuclease activity of, e.g., a TaqPolymerase such that the fluorescent emission of the donor fluorescentmoiety is no longer quenched. Exemplary probes for this purpose aredescribed in, e.g., U.S. Pat. Nos. 5,210,015, 5,994,056, and 6,171,785.Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonlyused quenchers are DABCYL and TAMRA. Commonly used dark quenchersinclude BlackHole Quenchers™ (BHQ) (such as BHQ2), (BiosearchTechnologies, Inc., Novato, Calif.), Iowa Black™, (Integrated DNA Tech.,Inc., Coralville, Iowa), BlackBerry™ Quencher 650 (BBQ-650), (Berry &Assoc., Dexter, Mich.).

In another example, two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the target nucleic acid sequence. Uponhybridization of the oligonucleotide probes to the amplification productnucleic acid at the appropriate positions, a FRET signal is generated.Hybridization temperatures can range from about 35° C. to about 65° C.for about 10 sec to about 1 min.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorimeter. Excitation to initiate energy transfer, or to allow directdetection of a fluorophore, can be carried out with an argon ion laser,a high intensity mercury (Hg) arc lamp, a xenon lamp, a fiber opticlight source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptor moieties“corresponding” refers to an acceptor fluorescent moiety or a darkquencher having an absorbance spectrum that overlaps the emissionspectrum of the donor fluorescent moiety. The wavelength maximum of theemission spectrum of the acceptor fluorescent moiety should be at least100 nm greater than the wavelength maximum of the excitation spectrum ofthe donor fluorescent moiety. Accordingly, efficient non-radiativeenergy transfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Foerster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,helium-cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine×isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm can be the distance in Angstroms (Å) from the nucleotide baseto the fluorescent moiety. In general, a linker arm is from about 10 Åto about 25 Å. The linker arm may be of the kind described inInternational Patent Publication No. WO 84/03285. International PatentPublication No. WO 84/03285 also discloses methods for attaching linkerarms to a particular nucleotide base, and also for attaching fluorescentmoieties to a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640, can be combinedwith an oligonucleotide that contains an amino linker (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or GlenResearch (Sterling, Va.)) to produce, for example, LC Red 640-labeledoligonucleotide. Frequently used linkers to couple a donor fluorescentmoiety such as fluorescein to an oligonucleotide include thiourealinkers (FITC-derived, for example, fluorescein-CPG's from Glen Researchor ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of Amplified Target Nucleic Acid Product (Amplicon)

The present disclosure provides methods for detecting and quantitatingtarget nucleic (including microbial target nucleic acids, such as HPV,WNV, HAV, HBV, HCV, and HIV) in a biological or non-biological sample.Methods provided avoid problems of sample contamination, falsenegatives, and false positives. The methods include performing at leastone cycling step that includes amplifying a portion of target nucleicacid molecules from a sample (e.g., HPV, WNV, HAV, HBV, HCV, or HIV)using one or more pairs of target primers, a non-extending helperoligonucleotide, and a FRET detecting step. Multiple cycling steps areperformed, preferably in a thermocycler. Methods can be performed usingthe target primers, non-extending helper oligonucleotides, and probes todetect the presence of a target nucleic acid (e.g., HPV, WNV, HAV, HBV,HCV, or HIV). Detection of the target nucleic acid (by, e.g., a probe)indicates the presence of the target nucleic acid (e.g., HPV, WNV, HAV,HBV, HCV, or HIV) in the sample.

As described herein, amplification products can be detected usinglabeled hybridization probes that take advantage of FRET technology. OneFRET format utilizes TaqMan® technology to detect the presence orabsence of an amplification product, and hence, the presence or absenceof HCV virus. TaqMan® technology utilizes one single-strandedhybridization probe labeled with, e.g., one fluorescent dye (e.g., HEX)and one quencher (e.g., BHQ or BHQ-2), which may or may not befluorescent. When a first fluorescent moiety is excited with light of asuitable wavelength, the absorbed energy is transferred to a secondfluorescent moiety or a dark quencher according to the principles ofFRET. The second moiety is generally a quencher molecule. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product) and isdegraded by the 5′ to 3′ nuclease activity of, e.g., the Taq Polymeraseduring the subsequent elongation phase. As a result, the fluorescentmoiety and the quencher moiety become spatially separated from oneanother. As a consequence, upon excitation of the first fluorescentmoiety in the absence of the quencher, the fluorescence emission fromthe first fluorescent moiety can be detected. By way of example, an ABIPRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan®technology, and is suitable for performing the methods described hereinfor detecting the presence or absence of a target nucleicacid/amplification product in the sample.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real-time PCRmethods. Molecular beacon technology uses a hybridization probe labeledwith a first fluorescent moiety and a second fluorescent moiety. Thesecond fluorescent moiety is generally a quencher, and the fluorescentlabels are typically located at each end of the probe. Molecular beacontechnology uses a probe oligonucleotide having sequences that permitsecondary structure formation (e.g., a hairpin). As a result ofsecondary structure formation within the probe, both fluorescentmoieties are in spatial proximity when the probe is in solution. Afterhybridization to the target nucleic acids (i.e., amplificationproducts), the secondary structure of the probe is disrupted and thefluorescent moieties become separated from one another such that afterexcitation with light of a suitable wavelength, the emission of thefirst fluorescent moiety can be detected.

Another common format of FRET technology utilizes two hybridizationprobes. Each probe can be labeled with a different fluorescent moietyand are generally designed to hybridize in close proximity to each otherin a target DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler® Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler® instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of HCV genomes). If amplification of targetnucleic acid occurs and an amplification product is produced, the stepof hybridizing results in a detectable signal based upon FRET betweenthe members of the pair of probes.

Generally, the presence of FRET indicates the presence of the targetnucleic acid in the sample, and the absence of FRET indicates theabsence of the target nucleic acid in the sample. Inadequate specimencollection, transportation delays, inappropriate transportationconditions, or use of certain collection swabs (calcium alginate oraluminum shaft) are all conditions that can affect the success and/oraccuracy of a test result, however.

Representative biological samples that can be used in practicing themethods include, but are not limited to respiratory specimens, urine,fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs,wound swabs, blood cultures, skin, and soft tissue infections.Collection and storage methods of biological samples are known to thoseof skill in the art. Biological samples can be processed (e.g., bynucleic acid extraction methods and/or kits known in the art) to releasethe target nucleic acid (microbial target nucleic acids, such as HPV,WNV, HAV, HBV, HCV, and HIV) or in some cases, the biological sample canbe contacted directly with the PCR reaction components and theappropriate oligonucleotides.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the target nucleic acid probes from the targetnucleic acid amplification products can confirm the presence or absenceof target nucleic acid in the sample (e.g., microbial target nucleicacids, such as HPV, WNV, HAV, HBV, HCV, and HIV).

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side-by-side with the patients' samples using the same primers andprobe as used for detection of the intended target. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Each thermocycler run can alsoinclude a negative control that, for example, lacks target template DNA.Negative control can measure contamination. This ensures that the systemand reagents would not give rise to a false positive signal. Therefore,control reactions can readily determine, for example, the ability ofprimers to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

In an embodiment, the methods include steps to avoid contamination. Forexample, an enzymatic method utilizing uracil-DNA glycosylase isdescribed in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduceor eliminate contamination between one thermocycler run and the next.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods. In one embodiment, a LightCycler® instrument isused. The following patent applications describe real-time PCR as usedin the LightCycler® technology: International Patent Publication Nos. WO97/46707, WO 97/46714, and WO 97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

One of skill in the art would appreciate that other nucleic acid- orsignal-amplification methods may also be employed. Examples of suchmethods include, without limitation, branched DNA signal amplification,loop-mediated isothermal amplification (LAMP), nucleic acidsequence-based amplification (NASBA), self-sustained sequencereplication (3 SR), strand displacement amplification (SDA), or smartamplification process version 2 (SMAP 2).

It is understood that the embodiments of the present disclosure are notlimited by the configuration of one or more commercially availableinstruments.

Articles of Manufacture/Kits

Embodiments of the present disclosure further provide for articles ofmanufacture or kits to detect and quantitate a target nucleic acid(e.g., microbial target nucleic acids, such as HPV, WNV, HAV, HBV, HCV,and HIV). An article of manufacture can include primers, non-extendinghelper oligonucleotides, and probes used to detect the gene target(e.g., microbial target nucleic acids, such as HPV, WNV, HAV, HBV, HCV,and HIV), together with suitable packaging materials. Representativeprimers and probes for detection and quantitation of a target nucleicacid (e.g., microbial target nucleic acids, such as HPV, WNV, HAV, HBV,HCV, and HIV) are capable of hybridizing to target nucleic acidmolecules. Representative non-extending helper oligonucleotides arecapable of annealing/hybridizing, in part or in whole, to the targetnucleic acid molecules. In some cases, the non-extending helperoligonucleotide and the (extending) primers anneal/hybridize to the sameportion, in part or in whole, of the target nucleic acid. That is, thenon-extending helper oligonucleotide may be designed in theprimer-binding region. Alternatively, the non-extending helperoligonucleotide and the (extending) primers do not anneal/hybridize tothe same portion of the target nucleic acid. In addition, the kits mayalso include suitably packaged reagents and materials needed for DNAimmobilization, hybridization, detection, quantitation, such as solidsupports, buffers, enzymes, and DNA standards. Methods of designingprimers, non-extending helper oligonucleotides, and probes are disclosedherein, and representative examples of primers, non-extending helperoligonucleotides, and probes that amplify and hybridize to targetnucleic acid molecules are provided.

Articles of manufacture can also include one or more fluorescentmoieties for labeling the probes or, alternatively, the probes suppliedwith the kit can be labeled. For example, an article of manufacture mayinclude a donor and/or an acceptor fluorescent moiety for labeling thetarget nucleic acid probes (e.g., probes specific for microbial targetnucleic acids, such as HPV, WNV, HAV, HBV, HCV, and HIV). Examples ofsuitable FRET donor fluorescent moieties and corresponding acceptorfluorescent moieties are provided above.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the primers, non-extendinghelper oligonucleotides, and probes to detect and quantitate targetnucleic acid (e.g., microbial target nucleic acids, such as HPV, WNV,HAV, HBV, HCV, and HIV) in a sample. Articles of manufacture mayadditionally include reagents for carrying out the methods disclosedherein (e.g., buffers, polymerase enzymes (e.g., DNA polymerase),nucleoside triphosphate monomers or other nucleoside monomers,co-factors, or agents to prevent contamination). Such reagents may bespecific for one of the commercially available instruments describedherein.

Embodiments of the present disclosure also provide for a set of primers,one or more non-extending helper oligonucleotides, and one or moredetectable probes for the detection of a target nucleic acid (e.g.,microbial target nucleic acids, such as HPV, WNV, HAV, HBV, HCV, andHIV) in a sample.

Reaction Mixtures

Embodiments of the present disclosure further provide for reactionmixtures to amplify, detect, and quantitate a target nucleic acid (e.g.,microbial target nucleic acids, such as HPV, WNV, HAV, HBV, HCV, andHIV). A reaction mixture can include primers, non-extending helperoligonucleotides, and probes used to detect the gene target (e.g.,microbial target nucleic acids, such as HPV, WNV, HAV, HBV, HCV, andHIV), together with suitable packaging materials. Representativeprimers, non-extending helper oligonucleotides, and probes for detectionand quantitation of a target nucleic acid (e.g., microbial targetnucleic acids, such as HPV, WNV, HAV, HBV, HCV, and HIV) are capable ofhybridizing to target nucleic acid molecules. In addition, the kits mayalso include suitably packaged reagents and materials needed for DNAimmobilization, hybridization, detection, quantitation, such as solidsupports, buffers, enzymes, and DNA standards. Methods of designingprimers, non-extending helper oligonucleotides, and probes are disclosedherein, and representative examples of primers, non-extending helperoligonucleotides, and probes that amplify and hybridize to targetnucleic acid molecules are provided.

Reaction mixtures can also include one or more fluorescent moieties forlabeling the probes or, alternatively, the probes supplied with the kitcan be labeled. For example, a reaction mixture may include a donorand/or an acceptor fluorescent moiety for labeling the target nucleicacid probes (e.g., probes specific for microbial target nucleic acids,such as HPV, WNV, HAV, HBV, HCV, and HIV). Examples of suitable FRETdonor fluorescent moieties and corresponding acceptor fluorescentmoieties are provided above.

Reaction mixtures may additionally include reagents for carrying out themethods disclosed herein (e.g., buffers, polymerase enzymes (e.g., DNApolymerase), nucleoside triphosphate monomers or other nucleosidemonomers, co-factors, or agents to prevent contamination). Such reagentsmay be specific for one of the commercially available instrumentsdescribed herein.

Embodiments of the present disclosure also provide for a set of primers,one or more non-extending helper oligonucleotides, and one or moredetectable probes for the detection of a target nucleic acid (e.g.,microbial target nucleic acids, such as HPV, WNV, HAV, HBV, HCV, andHIV) in a sample.

One embodiment of the invention is directed to a method for detectingand/or quantitating a target nucleic acid in a sample comprising: (a)contacting nucleic acids in the sample with amplification reagents, theamplification reagents comprising: at least an enzyme comprising DNApolymerase activity; at least nucleoside triphosphate monomers or othernucleoside monomers; at least one forward primer specific for the targetnucleic acid, or at least one reverse primer specific for the targetnucleic acid, or a combination thereof, for generating at least oneamplicon; at least one non-extending helper oligonucleotide, wherein:(i) the non-extending helper oligonucleotide does not extend, (ii) thenon-extending helper oligonucleotide anneals to part of the same portionof the target nucleic acid as at least one of the primers, and (iii) thenon-extending helper oligonucleotide enhances the activity of at leastone of the primers; and at least one detectable probe specific for theamplicon or at least one DNA binding dye; (b) incubating the nucleicacids with the amplification reagents for a period of time and underconditions sufficient for an amplification reaction to occur; and (c)detecting the amplicon with the at least one detectable probe or the atleast one DNA binding dye. In another embodiment, the target nucleicacid is a microbial nucleic acid (such as a viral nucleic acid and/or abacterial nucleic acid). In some embodiments, the viral nucleic acid isa nucleic acid of Human Papilloma Virus (HPV), West Nile Virus (WNV),Human Immunodeficiency Virus (HIV), Hepatitis A Virus (HAV), Hepatitis BVirus (HBV), or Hepatitis C Virus (HCV). In a related embodiment, theviral nucleic acid is a nucleic acid of Hepatitis C Virus (HCV),including, e.g., HCV Genotype 5. In another embodiment the at least onenon-extending helper oligonucleotide comprises a poly(A) sequence at the3′-end of the at least one non-extending helper oligonucleotide. In arelated embodiment, the poly(A) sequence is between 4-12 nucleotides inlength, such as about 8 nucleotides in length. In another embodiment,the at least one non-extending helper oligonucleotide comprises thesequence of SEQ ID NO:5, or a complementary sequence thereof. In anotherembodiment, the at least one forward primer comprises the sequence ofSEQ ID NO:1, or a complementary sequence thereof. In another embodiment,the at least one reverse primer comprises the sequence selected from thegroup consisting of SEQ ID NOs:2 and 3, or a complementary sequencethereof. In another embodiment, the at least one probe comprises thesequence of SEQ ID NO:4, or a complementary sequence thereof. In yetanother embodiment, the at least one forward primer comprises thesequence of SEQ ID NO:1, or a complementary sequence thereof; the atleast one reverse primer comprises the sequence selected from the groupconsisting of SEQ ID NOs:2 and 3, or a complementary sequence thereofand the at least one probe comprises the sequence of SEQ ID NO:4, or acomplementary sequence thereof.

Another embodiment of the present invention is directed to a method fordetecting and/or quantitating an HCV nucleic acid in a samplecomprising: (a) contacting nucleic acids in the sample withamplification reagents, the amplification reagents comprising: at leastan enzyme comprising DNA polymerase activity; at least nucleosidetriphosphate monomers or other nucleoside monomers; at least one forwardprimer specific for the target nucleic acid, or at least one reverseprimer specific for the target nucleic acid, or a combination thereof,for generating at least one amplicon; at least one non-extending helperoligonucleotide, wherein: (i) the non-extending helper oligonucleotidedoes not extend, (ii) the non-extending helper oligonucleotide annealsto part of the same portion of the target nucleic acid as at least oneof the primers, and (iii) the non-extending helper oligonucleotideenhances the activity of at least one of the primers; and at least onedetectable probe specific for the amplicon or at least one DNA bindingdye; (b) incubating the nucleic acids with the amplification reagentsfor a period of time and under conditions sufficient for anamplification reaction to occur; and (c) detecting the amplicon with theat least one detectable probe or the at least one DNA binding dye. Inanother embodiment, the at least one non-extending helperoligonucleotide comprises a poly(A) sequence at the 3′-end of the atleast one non-extending helper oligonucleotide. In another embodiment,the poly(A) sequence is between 4-12 nucleotides in length, for example,about 8 nucleotides in length. In another embodiment, the HCV is HCVGenotype 5. In another embodiment, the at least one non-extending helperoligonucleotide comprises the sequence of SEQ ID NO:5, or acomplementary sequence thereof. In a related embodiment, the at leastone forward primer comprises the sequence of SEQ ID NO:1, or acomplementary sequence thereof. In another embodiment, the at least onereverse primer comprises the sequence selected from the group consistingof SEQ ID NOs:2 and 3, or a complementary sequence thereof. In anotherembodiment, the at least one probe comprises the sequence of SEQ IDNO:4, or a complementary sequence thereof. In another embodiment, the atleast one forward primer comprises the sequence of SEQ ID NO:1, or acomplementary sequence thereof; the at least one reverse primercomprises the sequence selected from the group consisting of SEQ IDNOs:2 and/or 3, or a complementary sequence thereof; and the at leastone probe comprises the sequence of SEQ ID NO:4, or a complementarysequence thereof.

Another embodiment of the present invention is directed to a kit foramplifying and detecting and/or quantitating a target nucleic acid in asample comprising amplification reagents, the amplification reagentscomprising: (a) an enzyme comprising DNA polymerase activity; (b)nucleoside triphosphate monomers or other nucleoside monomers; (c) atleast one forward primer or at least one reverse primer specific for thetarget nucleic acid, or a combination thereof; (d) at least onenon-extending helper oligonucleotide, wherein: (i) the non-extendinghelper oligonucleotide does not extend, (ii) the non-extending helperoligonucleotide anneals to part of the same portion of the targetnucleic acid as at least one of the primers, and (iii) the non-extendinghelper oligonucleotide enhances the activity of at least one of theprimers; and (e) at least one detectable probe for the target nucleicacid or a DNA binding dye. In another embodiment, the target nucleicacid is a microbial nucleic acid (such as a viral nucleic acid or abacterial nucleic acid). In a related embodiment, the viral nucleic acidis a nucleic acid of HPV, WNV, HIV, HAV, HBV, or HCV. In a moreparticular embodiment, the viral nucleic acid is a nucleic acid of HCV,including, for example, HCV Genotype 5. In another embodiment, the atleast one non-extending helper oligonucleotide comprises a poly(A)sequence at the 3′-end of the at least one non-extending helperoligonucleotide. In another embodiment, the poly(A) sequence is between4-12 nucleotides in length, for example, about 8 nucleotides in length.In another embodiment, the at least one non-extending helperoligonucleotide comprises the sequence of SEQ ID NO:5, or acomplementary sequence thereof. In another embodiment, the at least oneforward primer comprises the sequence of SEQ ID NO:1, or a complementarysequence thereof. In another embodiment, the at least one reverse primercomprises the sequence selected from the group consisting of SEQ IDNOs:2 and 3, or a complementary sequence thereof. In another embodiment,the at least one probe comprises the sequence of SEQ ID NO:4, or acomplementary sequence thereof. In another embodiment, the at least oneforward primer comprises the sequence of SEQ ID NO:1, or a complementarysequence thereof; the at least one reverse primer comprises the sequenceselected from the group consisting of SEQ ID NOs:2 and 3, or acomplementary sequence thereof; and the at least one probe comprises thesequence of SEQ ID NO:4, or a complementary sequence thereof.

A related embodiment of the present invention is directed to a kit foramplifying and detecting and/or quantitating a HCV nucleic acid in asample comprising amplification reagents, the amplification reagentscomprising: (a) an enzyme comprising DNA polymerase activity; (b)nucleoside triphosphate monomers or other nucleoside monomers; (c) atleast one forward primer or at least one reverse primer specific for thetarget nucleic acid, or a combination thereof; (d) at least onenon-extending helper oligonucleotide, wherein: (i) the non-extendinghelper oligonucleotide does not extend, (ii) the non-extending helperoligonucleotide anneals to part of the same portion of the targetnucleic acid as at least one of the primers, and (iii) the non-extendinghelper oligonucleotide enhances the activity of at least one of theprimers; and (e) at least one detectable probe for the target nucleicacid or a DNA binding dye. In another embodiment, the at least onenon-extending helper oligonucleotide comprises a poly(A) sequence at the3′-end of the at least one non-extending helper oligonucleotide. Inanother embodiment, the poly(A) sequence is between 4-12 nucleotides inlength, for example, about 8 nucleotides in length. In anotherembodiment, the HCV is HCV Genotype 5. In another embodiment, the atleast one non-extending helper oligonucleotide comprises the sequence ofSEQ ID NO:5, or a complementary sequence thereof. In a relatedembodiment, the at least one forward primer comprises the sequence ofSEQ ID NO:1, or a complementary sequence thereof. In another embodiment,the at least one reverse primer comprises the sequence selected from thegroup consisting of SEQ ID NOs:2 and 3, or a complementary sequencesthereof. In another embodiment, the at least one probe comprises thesequence of SEQ ID NO:4, or a complementary sequence thereof. In anotherembodiment, the at least one forward primer comprises the sequence ofSEQ ID NO:1, or a complementary sequence thereof the at least onereverse primer comprises the sequence selected from the group consistingof SEQ ID NOs:2 and 3, or a complementary sequence thereof and the atleast one probe comprises the sequence of SEQ ID NO:4, or acomplementary sequence thereof.

Yet another embodiment of the present invention is directed to areaction mixture effective to amplify and detect and/or quantitate atarget nucleic acid in a sample comprising amplification reagentscomprising: (a) a sample; (b) an enzyme comprising DNA polymeraseactivity; (c) nucleoside triphosphate monomers or other nucleosidemonomers; (d) at least one forward primer or at least one reverse primerspecific for the target nucleic acid, or a combination thereof (e) atleast one non-extending helper oligonucleotide, wherein: (i) thenon-extending helper oligonucleotide does not extend, (ii) thenon-extending helper oligonucleotide anneals to part of the same portionof the target nucleic acid as at least one of the primers, and (iii) thenon-extending helper oligonucleotide enhances the activity of at leastone of the primers; and (f) at least one detectable probe for the targetnucleic acid or a DNA binding dye. In another embodiment, the targetnucleic acid is a microbial nucleic acid. In another embodiment, themicrobial nucleic acid is a viral nucleic acid. In another embodiment,the microbial nucleic acid is a bacterial nucleic acid. In anotherembodiment, the viral nucleic acid is a nucleic acid of HPV, WNV, HIV,HAV, HBV, or HCV. In a particular embodiment, the viral nucleic acid isa nucleic acid of HCV, including, for example, HCV Genotype 5. Inanother embodiment, the at least one non-extending helperoligonucleotide comprises a poly(A) sequence at the 3′-end of the atleast one non-extending helper oligonucleotide. In another embodiment,the poly(A) sequence is between 4-12 nucleotides in length, for example,about 8 nucleotides in length. In another embodiment, the at least onenon-extending helper oligonucleotide comprises the sequence of SEQ IDNO:5, or a complementary sequence thereof. In another embodiment, the atleast one forward primer comprises the sequence of SEQ ID NO:1, or acomplementary sequence thereof. In another embodiment, the at least onereverse primer comprises the sequence selected from the group consistingof SEQ ID NOs:2 and 3, or a complementary sequence thereof. In anotherembodiment, the at least one probe comprises the sequence of SEQ IDNO:4, or a complementary sequence thereof. In another embodiment, the atleast one forward primer comprises the sequence of SEQ ID NO:1, or acomplementary sequence thereof the at least one reverse primer comprisesthe sequence selected from the group consisting of SEQ ID NOs:2 and 3,or a complementary sequence thereof; and the at least one probecomprises the sequence of SEQ ID NO:4, or a complementary sequencethereof.

A related embodiment of the present invention is directed to a reactionmixture effective to amplify and detect and/or quantitate a HCV nucleicacid in a sample, comprising amplification reagents comprising: (a) asample; (b) an enzyme comprising DNA polymerase activity; (c) nucleosidetriphosphate monomers or other nucleoside monomers; (d) at least oneforward primer or at least one reverse primer specific for the targetnucleic acid, or a combination thereof; (e) at least one non-extendinghelper oligonucleotide, wherein: (i) the non-extending helperoligonucleotide does not extend, (ii) the non-extending helperoligonucleotide anneals to part of the same portion of the targetnucleic acid as at least one of the primers, and (iii) the non-extendinghelper oligonucleotide enhances the activity of at least one of theprimers; and (f) at least one detectable probe for the target nucleicacid or a DNA binding dye. In another embodiment, the at least onenon-extending helper oligonucleotide comprises a poly(A) sequence at the3′-end of the at least one non-extending helper oligonucleotide. Inanother embodiment, the poly(A) sequence is between 4-12 nucleotides inlength, for example about 8 nucleotides in length. In one embodiment,the HCV is HCV Genotype 5. In another embodiment, the at least onenon-extending helper oligonucleotide comprises the sequence of SEQ IDNO:5, or a complementary sequence thereof. In another embodiment, the atleast one forward primer comprises the sequence of SEQ ID NO:1, or acomplementary sequence thereof. In another embodiment, the at least onereverse primer comprises the sequence selected from the group consistingof SEQ ID NOs:2 and 3, or a complementary sequences thereof. In anotherembodiment, the at least one probe comprises the sequence of SEQ IDNO:4, or a complementary sequence thereof. In another embodiment, the atleast one forward primer comprises the sequence of SEQ ID NO:1, or acomplementary sequence thereof the at least one reverse primer comprisesthe sequence selected from the group consisting of SEQ ID NOs:2 and 3,or a complementary sequence thereof and the at least one probe comprisesthe sequence of SEQ ID NO:4, or a complementary sequence thereof.

Embodiments of the present disclosure will be further described in thefollowing examples, which do not limit the scope of the inventiondescribed in the claims.

EXAMPLES

The following examples and figures are provided to aid the understandingof the subject matter, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example 1: Detection of HCV by Real-Time PCR

RNA samples used for a real-time PCR assay were extracted from aHCV-positive human plasma sample. Reagents used include Cobas® 6800/8800generic PCR Master Mix, with the profile and conditions for use with theCobas® 6800/8800, and using TaqMan® detection and quantitationtechnology. In 50 μl of PCR reaction, the final concentrations were asfollows: forward and reverse primers ranged from 0.2-0.5 μM;non-extending helper oligonucleotide was at 0.20 μM; and probe was at0.12 μM. It is believed that the forward primer, reverse primers, andnon-extending helper oligonucleotides at a concentration within a rangeof 0.10 μM-0.50 μM would be effective.

To demonstrate the effect of the non-extending helper oligonucleotide, areal-time PCR assay using HCV-positive human plasma samples wasconducted in duplicate with the following two experimental conditions:(1) real-time PCR with primer pairs and probe only (i.e., withoutnon-extending helper oligonucleotide); and (2) real-time PCR with primerpairs, non-extending helper oligonucleotide, and probe. Theoligonucleotides specific for HCV Genotype 5 used for the real-time PCRassay were SEQ ID NO:1 for the forward primer, SEQ ID NOs:2 and 3 forthe reverse primers, SEQ ID NO:5 for the non-extending helperoligonucleotide, and SEQ ID NO:4 for the probe. The non-extending helperoligonucleotide (SEQ ID NO:5) is a 53 base pair unlabeledoligonucleotide with a poly(A) tail at its 3′-end and is designed in thereverse primer binding core region of the HCV genome.

For this these HCV-positive human plasma samples, the predictedsecondary structure of the HCV target region in the presence of thenon-extending helper oligonucleotide is depicted in FIG. 2, showing a ΔGof −107.2 kcal/mol (CLC Genomics Workbench 6, Qiagen), and the resultsare shown in FIG. 1, which shows real-time PCR growth curves. As can beseen in FIG. 1, the PCR reaction is more efficient in the presence of anon-extending helper oligonucleotide than without

These results demonstrate that the PCR reaction is more robust and moreefficient in the presence of the non-extended helper oligonucleotide.That is, there is improved accumulation of the PCR product in thepresence of the non-extended helper oligonucleotide than without

Example 2: Detection of HCV Genotype 5 by Real-Time PCR

As in Example 1, RNA samples used for a real-time PCR assay, but wereextracted from a different HCV-positive human plasma sample than thesample used in Example 1. As in Example, 1, reagents used include Cobas®6800/8800 generic PCR Master Mix, with the profile and conditions foruse with the Cobas® 6800/8800, and using TaqMan® detection andquantitation technology. In 50 μl of PCR reaction, the finalconcentrations were as follows: forward and reverse primers ranged from0.2-0.5 μM; non-extending helper oligonucleotide was at 0.20 μM; andprobe was at 0.12 μM. It is believed that the forward primer, reverseprimers, and non-extending helper oligonucleotides at a concentrationwithin a range of 0.10 μM-0.50 μM would be effective.

To demonstrate the effect of the non-extending helper oligonucleotide, areal-time PCR assay using HCV-positive human plasma samples (differentfrom the samples employed in Example 1) was conducted in duplicate withthe following two experimental conditions: (1) real-time PCR with primerpairs and probe only (i.e., without non-extending helperoligonucleotide); and (2) real-time PCR with primer pairs, non-extendinghelper oligonucleotide, and probe. The oligonucleotides specific for HCVGenotype 5 used for the real-time PCR assay were SEQ ID NO:1 for theforward primer, SEQ ID NOs:2 and 3 for the reverse primers, SEQ ID NO:5for the non-extending helper oligonucleotide, and SEQ ID NO:4 for theprobe. As in Example 1, the non-extending helper oligonucleotide (SEQ IDNO:5) is a 53 base pair unlabeled oligonucleotide with a poly(A) tail atits 3′-end and is designed in the reverse primer binding core region ofthe HCV genome.

For this particular HCV-positive human plasma sample, the predictedsecondary structure of the HCV target region in the presence of thenon-extending helper oligonucleotide is depicted in FIG. 4, showing a ΔGof −98.6 kcal/mol and the results are shown in FIG. 3, which showsreal-time PCR growth curves. As can be seen in FIG. 3, the PCR reactionis more efficient in the presence of a non-extending helperoligonucleotide than without.

These results demonstrate that the PCR reaction is more robust and moreefficient in the presence of the non-extended helper oligonucleotide.That is, there is improved accumulation of the PCR product in thepresence of the non-extended helper oligonucleotide than without.

Taken together, these two examples demonstrate that in differentsamples, the non-extending helper oligonucleotide of the presentinvention appears to lower the Gibbs Free Energy of the secondarystructure of the target nucleic acid, which facilitates access of thetarget nucleic acid to the primers. This results in an improvement inamplification efficiency. Thus, the non-extending helper oligonucleotideincreases the sensitivity of an amplification assay.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1. A method for detecting and/or quantitating a target nucleic acid in asample comprising: (a) contacting nucleic acids in the sample withamplification reagents, the amplification reagents comprising: at leastan enzyme comprising DNA polymerase activity; at least nucleosidetriphosphate monomers or other nucleoside monomers; at least one forwardprimer specific for the target nucleic acid, or at least one reverseprimer specific for the target nucleic acid, or a combination thereof,for generating at least one amplicon; at least one non-extending helperoligonucleotide, wherein: (i) the non-extending helper oligonucleotidedoes not extend, (ii) the non-extending helper oligonucleotide annealsto part of the same portion of the target nucleic acid as at least oneof the primers, and (iii) the non-extending helper oligonucleotideenhances the activity of at least one of the primers; and at least onedetectable probe specific for the amplicon or at least one DNA bindingdye; (b) incubating the nucleic acids with the amplification reagentsfor a period of time and under conditions sufficient for anamplification reaction to occur; and (c) detecting the amplicon with theat least one detectable probe or the at least one DNA binding dye. 2.The method of claim 1, wherein the target nucleic acid is a microbialnucleic acid.
 3. The method of claim 2, wherein the microbial nucleicacid is a bacterial nucleic acid.
 4. The method of claim 2, wherein themicrobial nucleic acid is a viral nucleic acid.
 5. The method of claim4, wherein the viral nucleic acid is a nucleic acid of Human PapillomaVirus (HPV), West Nile Virus (WNV), Human Immunodeficiency Virus (HIV),Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), or Hepatitis C Virus(HCV).
 6. The method of claim 5, wherein the viral nucleic acid is anucleic acid of HCV.
 7. The method of claim 6, wherein the HCV is HCVGenotype
 5. 8. The method of any one of claims 1-7, wherein the at leastone non-extending helper oligonucleotide comprises a poly(A) sequence atthe 3′-end of the at least one non-extending helper oligonucleotide. 9.The method of claim 8, wherein the poly(A) sequence is between 4-12nucleotides in length.
 10. (canceled)
 11. The method of claim 7, whereinthe at least one non-extending helper oligonucleotide comprises thesequence of SEQ ID NO:5, or a complementary sequence thereof. 12.(canceled)
 13. (canceled)
 14. (canceled)
 15. The method of claim 11,wherein the at least one forward primer comprises the sequence of SEQ IDNO:1, or a complementary sequence thereof the at least one reverseprimer comprises the sequence selected from the group consisting of SEQID NOs:2 and 3, or a complementary sequence thereof; and the at leastone probe comprises the sequence of SEQ ID NO:4, or a complementarysequence thereof.
 16. A method for detecting and/or quantitating an HCVnucleic acid in a sample comprising: (a) contacting nucleic acids in thesample with amplification reagents, the amplification reagentscomprising: at least an enzyme comprising DNA polymerase activity; atleast nucleoside triphosphate monomers or other nucleoside monomers; atleast one forward primer specific for the target nucleic acid, or atleast one reverse primer specific for the target nucleic acid, or acombination thereof, for generating at least one amplicon; at least onenon-extending helper oligonucleotide, wherein: (i) the non-extendinghelper oligonucleotide does not extend, (ii) the non-extending helperoligonucleotide anneals to part of the same portion of the targetnucleic acid as at least one of the primers, and (iii) the non-extendinghelper oligonucleotide enhances the activity of at least one of theprimers; and at least one detectable probe specific for the amplicon orat least one DNA binding dye; (b) incubating the nucleic acids with theamplification reagents for a period of time and under conditionssufficient for an amplification reaction to occur; and (c) detecting theamplicon with the at least one detectable probe or the at least one DNAbinding dye.
 17. The method of claim 16, wherein the at least onenon-extending helper oligonucleotide comprises a poly(A) sequence at the3′-end of the at least one non-extending helper oligonucleotide.
 18. Themethod of claim 17, wherein the poly(A) sequence is between 4-12nucleotides in length.
 19. (canceled)
 20. The method of claim 16,wherein the HCV is HCV Genotype
 5. 21. The method of claim 20, whereinthe at least one non-extending helper oligonucleotide comprises thesequence of SEQ ID NO:5, or a complementary sequence thereof. 22.(canceled)
 23. (canceled)
 24. (canceled)
 25. The method of claim 21,wherein the at least one forward primer comprises the sequence of SEQ IDNO:1, or a complementary sequence thereof; the at least one reverseprimer comprises the sequence selected from the group consisting of SEQID NOs:2 and/or 3, or a complementary sequence thereof; and the at leastone probe comprises the sequence of SEQ ID NO:4, or a complementarysequence thereof.
 26. A kit for amplifying and detecting and/orquantitating a target nucleic acid in a sample comprising amplificationreagents, the amplification reagents comprising: (a) an enzymecomprising DNA polymerase activity; (b) nucleoside triphosphate monomersor other nucleoside monomers; (c) at least one forward primer or atleast one reverse primer specific for the target nucleic acid, or acombination thereof; (d) at least one non-extending helperoligonucleotide, wherein: (i) the non-extending helper oligonucleotidedoes not extend, (ii) the non-extending helper oligonucleotide annealsto part of the same portion of the target nucleic acid as at least oneof the primers, and (iii) the non-extending helper oligonucleotideenhances the activity of at least one of the primers; and (e) at leastone detectable probe for the target nucleic acid or a DNA binding dye.27. The kit of claim 26, wherein the target nucleic acid is a microbialnucleic acid.
 28. The kit of claim 27, wherein the microbial nucleicacid is a bacterial nucleic acid.
 29. The kit of claim 27, wherein themicrobial nucleic acid is a viral nucleic acid.
 30. The kit of claim 29,wherein the viral nucleic acid is a nucleic acid of HPV, WNV, HIV, HAV,HBV, or HCV.
 31. The kit of claim 30, wherein the viral nucleic acid isa nucleic acid of HCV.
 32. The kit of claim 31, wherein the HCV is HCVGenotype
 5. 33. The kit of any one of claims 26-32, wherein the at leastone non-extending helper oligonucleotide comprises a poly(A) sequence atthe 3′-end of the at least one non-extending helper oligonucleotide. 34.The kit of claim 33, wherein the poly(A) sequence is between 4-12nucleotides in length.
 35. (canceled)
 36. The kit of claim 32, whereinthe at least one non-extending helper oligonucleotide comprises thesequence of SEQ ID NO:5, or a complementary sequence thereof. 37.(canceled)
 38. (canceled)
 39. (canceled)
 40. The kit of claim 36,wherein the at least one forward primer comprises the sequence of SEQ IDNO:1, or a complementary sequence thereof; the at least one reverseprimer comprises the sequence selected from the group consisting of SEQID NOs:2 and 3, or a complementary sequence thereof; and the at leastone probe comprises the sequence of SEQ ID NO:4, or a complementarysequence thereof.
 41. A reaction mixture effective to amplify and detectand/or quantitate a target nucleic acid in a sample comprisingamplification reagents comprising: (a) a sample; (b) an enzymecomprising DNA polymerase activity; (c) nucleoside triphosphate monomersor other nucleoside monomers; (d) at least one forward primer or atleast one reverse primer specific for the target nucleic acid, or acombination thereof; (e) at least one non-extending helperoligonucleotide, wherein: (i) the non-extending helper oligonucleotidedoes not extend, (ii) the non-extending helper oligonucleotide annealsto part of the same portion of the target nucleic acid as at least oneof the primers, and (iii) the non-extending helper oligonucleotideenhances the activity of at least one of the primers; and at least onedetectable probe for the target nucleic acid or a DNA binding dye. 42.The reaction mixture of claim 41, wherein the target nucleic acid is amicrobial nucleic acid.
 43. The reaction mixture of claim 42, whereinthe microbial nucleic acid is a bacterial nucleic acid.
 44. The reactionmixture of claim 42, wherein the microbial nucleic acid is a viralnucleic acid.
 45. The reaction mixture of claim 44, wherein the viralnucleic acid is a nucleic acid of HPV, WNV, HIV, HAV, HBV, or HCV. 46.The reaction mixture of claim 45, wherein the viral nucleic acid is anucleic acid of HCV.
 47. The reaction mixture of claim 46, wherein theHCV is HCV Genotype
 5. 48. The reaction mixture of claim 41, wherein theat least one non-extending helper oligonucleotide comprises a poly(A)sequence at the 3′-end of the at least one non-extending helperoligonucleotide.
 49. The reaction mixture of claim 48, wherein thepoly(A) sequence is between 4-12 nucleotides in length.
 50. (canceled)51. The reaction mixture of claim 49, wherein the at least onenon-extending helper oligonucleotide comprises the sequence of SEQ IDNO:5, or a complementary sequence thereof.
 52. (canceled)
 53. (canceled)54. (canceled)
 55. The reaction mixture of claim 51, wherein the atleast one forward primer comprises the sequence of SEQ ID NO:1, or acomplementary sequence thereof; the at least one reverse primercomprises the sequence selected from the group consisting of SEQ IDNOs:2 and 3, or a complementary sequence thereof; and the at least oneprobe comprises the sequence of SEQ ID NO:4, or a complementary sequencethereof.